An assay is defined as a test that identifies a chemical species, determines its presence, and can measure the amount of chemical specie present. The chemical specie is termed an “analyte” in the field of chemical analysis. These assays are relevant to many diagnostic situations.
Cyanide and some of the related species represent a class of very toxic substances. Cyanide and some of the related species are used for a class of compounds known as chemical warfare agents. It would be particularly valuable to be able to detect these chemical warfare agents at concentrations well below the toxic level in order to provide an early warning to potentially affected populations. Cyanide and related species also find widespread use in the mining and chemical manufacturing industries.
The SERS affect stems from an electromagnetic and often chemical enhancement of Raman scattering at a metal surface. Most often the metal surface is composed of silver or gold. These metals produce strong enhancements only under special conditions. One of these conditions is the size of the metal surface. A common method of performing SERS spectroscopy is to use the surface of small particles, with dimensions on the order of 1 to about 100 nanometers, as the SERS active media for analysis. These nanoparticles can produce large enhancements of the Raman scattering of an analyte when that analyte binds to the surface. The frequency at which one or several Raman bands is observed may be used to identify the chemical composition of the analyte and to distinguish it from other analytes that may bind to the SERS active surface. The intensity of the Raman band may be related to the amount of analyte present. The SERS effect has a very unique capability for detecting only species at or very near to the active surface. This means that properly constructed assays can proximally eliminate interferences from other species.
Ten to twenty years ago a SERS assay would most likely have been considered impractical because the instrumentation for Raman spectroscopy was very large, delicate, and expensive. Recent advances in optics, detectors, and lasers have made Raman systems small and compact enough for practical low-cost assays.
Modern methods of chemical analysis include wet chemistry, spectroscopy, chromatography, and electrochemistry. All of these methods vary in the amount of information that can be obtained in an analysis. For example, most electrochemical techniques measure current at a given applied potential. Such a technique would be considered poor in information content. Many simple spectroscopic measurements are also made by measuring an optical signal at a single wavelength. For example, ultraviolet or visible absorption spectroscopy is often performed with a low-cost instrument that illuminates the sample with a small band of wavelengths and the sample is quantitated using the linear relationship between the amount of light absorbed and the concentration. This too would constitute a technique that is poor in information content.
Cyanide analysis represents a good example of the problems with wet chemistry and electrochemical detection. Currently, the most popular method for cyanide measurement is a wet chemical method. It involves a dye that changes color when silver ions are present. Silver ions also react strongly with cyanide. The assay uses a solution with a known silver ion concentration to titrate an unknown sample of cyanide. After all silver ions in the known solution of silver ions have been complexed with cyanide, the presence of free silver ions is indicated by a color change due to the dye. This method can produce a fairly accurate analysis, but requires a significant amount of time to measure the cyanide concentration. Moreover, is highly subjective with regards to the color change, if performed by a person. It is also subject to severe interference from solutions that contain other species with complex with silver ions. Cyanide is electrochemically active and can be detected in pure solutions by electrochemistry. However, as electrochemistry is very sensitive to contamination and does not provide a distinguishing signal between cyanide and interferences, it is subject to error.
Also, in the case of cyanide, the desired quantity is often “free” cyanide. This is cyanide that is not bound to a metal. Free cyanide is most accurately measured by finding the “total” cyanide obtained by mixing the sample with a strong acid and collecting the evolved hydrogen cyanide gas in a sodium hydroxide solution and measuring the cyanide concentration in solution using the titration method described earlier. Next, a second measurement of the “wad” or weak acid dissociable cyanide is made by adding a weak acid to the sample and measuring the amount of free cyanide using the above titration method. The “free” cyanide in the sample is calculated by determining the difference between the total cyanide and the wad cyanide. The total procedure is very time consuming, labor intensive, and uses dangerous reagents.
Cyanide in blood is also very difficult to measure. It binds to the hemoglobin in the blood and is also present as “free” cyanide in the serum. A common method for measuring cyanide in blood is to add a strong acid to produce hydrogen cyanide and to detect the hydrogen cyanide with a gas chromatograph. Given that death from cyanide poisoning can occur within minutes, such an analysis is not practical for diagnosis.
As demonstrated above, current methods for measuring cyanide are time consuming and often require several steps to produce a meaningful value. For first responders, such as emergency medical technicians, a rapid cyanide analysis is needed. Moreover, in view of the recent concerns about chemical warfare, it would be even more advantageous to detect and prevent contact with cyanide before a blood analysis is needed.
Some modern methods of analysis are rich in information and provide rapid results. For example, spectroscopy based on molecular vibrations tend to produce data that is composed of a signal spread over a large range of wavelengths. This allows one to delineate the contribution of many analytes in the sample in a single spectrum. Two basic forms of vibrational spectroscopy exist: infrared absorption and Raman scattering. Infrared absorption spectroscopy measures the absorption of infrared radiation by molecules. The absorption occurs whenever the energy of the radiation matches the energy of a molecular vibration in the sample. Infrared absorption spectroscopy is rich in information, but can be difficult to use as an analytical tool. The infrared radiation is strongly absorbed by glass and other common optical materials. This requires the samples to be contained in materials like potassium bromide, which is very brittle and hard to shape into even the simplest sample container. This alone is a difficulty, but for quantitative analysis it is very difficult to control the thickness of the sample. If the thickness is unknown it is impossible to relate the absorbance to the concentration. Water is also a strong absorber of infrared radiation and interferes with analysis. This is particularly troublesome with aqueous samples.
Raman spectroscopy stems from the inelastic scattering of light by molecular vibrational energy levels. Also termed Normal Raman scattering, it is performed by exciting the sample with a strong optical source, usually a laser. The Raman scattered light is emitted from the sample and collected with an optical element, usually a lens. Once collected, the light is dispersed with a spectrometer and analyzed with an optical transducer. Raman spectroscopy, as an analytical tool, has been known for decades, and is particularly popular for several reasons. For example, molecular composition can be determined in the presence of water. Visible light can be employed for analysis allowing for the use of conventional optical materials. Unique spectral fingerprints allow for identification and quantification of a wide variety of solids, liquids, and gases. These advantages are overshadowed by an inherent lack of sensitivity of Raman scattering. This lack of sensitivity has precluded the use of Raman spectroscopy in application where low levels of material need to be detected, however SERS spectroscopy provides the means whereby it may be used in just such applications as described herein.
Surface enhanced Raman scattering (SERS) like many scientific discoveries, evolved out of serendipitous events. In the early 1970's, electrochemists began using optical methods to study electrode surfaces. Fleischmann and Hendra decided to experiment with Raman spectroscopy as a method of analyzing electrode surfaces. Due to the low sensitivity of Raman spectroscopy they chose silver as the electrode material since it is easily roughened by oxidation-reduction cycles in the presence of chloride. The growth of silver chloride crystals and reduction back to silver leads to a roughened surface with many times the surface area of a smooth polished electrode. This will increase the Raman signal, as there are more molecules in the laser beam. They chose pyridine as the probe molecule as it should adsorb through the pyridine nitrogen and it is an inherently strong Raman scatterer. Their experiment was a success. They did not know it, but this was the first experiment using SERS. It was not until four years later that this experiment was correctly interpreted. In 1977, Van Duyne at Northwestern University was also trying to study electrodes with Raman spectroscopy. His approach was to use resonantly enhanced molecular probes to overcome the sensitivity problem. Resonance Raman is an enhancement of Raman scattering achieved by exciting the molecule at a wavelength that matches an electronic absorption of the molecule. He had performed calculations to determine the amount of resonance enhancement needed to observe a monolayer on an electrode. This number was at least 1000 for a strong scatterer like pyridine. This made Flieschmann and Hendra's results look anomalous. To test if the enhancement was due to increased surface roughness, Van Duyne's student David Jeanmaire tried a milder oxidation-reduction cycle and achieved even stronger signals. This led to the first announcement of an anomalous phenomenon at silver surfaces.
It is now known that the SERS effect arises through an electromagnetic resonance that can occur strongly in noble metal particles and to a lesser extent in some other metals. The resonance occurs because the electrons in the particle are affected by the excitation light to produce a polarization in the particle that makes it more likely to become more polarized. This phenomenon will produce very large electric fields near the particle surface, thus amplifying optical events near the surface that are dependent on the electromagnetic field. Raman scattering is just one class of such events. Others might include fluorescence and absorbance.
While SERS was discovered on electrode surfaces, it is not limited to these. Today SERS is being performed on evaporated metal surfaces, etched metal foils, microlithographically produced surfaces, carefully assembled particle arrays, and colloidal suspensions. Other methods, capable of producing small submicron sized particles or features on a surface, also provide various SERS active surfaces.
Several problems have plagued the development of SERS into a practical analytical tool. One such problem is the delicate nature of the SERS substrate. The SERS phenomenon is associated with particles or roughness features that are about 1/10 the size of the wavelength of the light used for excitation or about 40 to 100 nanometers. Particles of this size are very susceptible to chemical damage, aggregation, and photodamage.
A survey of the different SERS substrates produces one type that stands out with respect to practical analytical chemistry. These are colloidal suspensions. Two significant advantages are found with colloidal suspensions. First, a large volume of colloidal particles can be made at one time. Within this batch of colloids, every sample will be identical. This overcomes the irreproducibility of non-free floating particulate surfaces. The second advantage is that the colloidal particles are suspended in a solution and therefore tend to be much less susceptible to thermal damage. They also are subject to Brownian motion, which tends to continually refresh the particles in the excitation beam, thus eliminating problems with photodegradation of the sample.
Initially SERS was seen as advantageous because of its strong enhancement. This invention realizes a different aspect of SERS. The localization of the SERS enhancement near the surface very effectively separates the analyte that is in close proximity with the surface from analyte or other material in the sample matrix. The locality of the analyte can be used to a strong advantage with respect to the ease of analysis. SERS allows one to measure an analyte in the presence of species that would strongly interfere and cripple other methods of analysis that do not have a localized area of detection.
In addition to problems with SERS substrate stability and reproducibility, an additional factor needs to be included in the analysis. The SERS substrates are typically noble metal particles. The noble metals are aptly named for their ability to resist the aggressions of other materials. In a practical sense this is good for stability of the surfaces, but is impractical in terms of attracting an analyte to the surface. In order for the SERS substrate to act as a tool for detecting an analyte, it must attract the analyte to the surface or in some way be specifically affected by the analyte to show a spectroscopic response.
Small nanoparticles of gold and silver react with some chemical species to create a strongly bound surface complex. This complex may be observed spectroscopically as a bound species. A condition for the observation of the bound species is the ability to distinguish between the surface-bound species and solution species or other interferences. This distinction often requires both a selectivity aspect related to the relationship between the energy of the light affected by the spectroscopic measurement and the intensity of the light affecting the spectroscopic measurement.
With respect to cyanide and related species, the noble metals gold and silver tend to form very strong complexes with these species. In solution or in the air, particles of silver or gold tend to bind cyanide very strongly to give a surface coating composed of metal cyanide complexes.
One example of a spectroscopic technique is Raman spectroscopy, which is very specific in its ability to measure between complexed and solution species or differences between cyanide species. Moreover, SERS is very surface specific, such that it can identify between solution and surface bound species.
The SERS phenomenon is electromagnetic and at times may also be due to the formation of a surface bound chemical species that is spectroscopically unique and distinguishes itself to provide an indication of the presence of the surface species. For example, the formation of a complex that has an electronic absorption may lend itself to detection through UV-Vis absorption spectroscopy, fluorescence, or resonance Raman spectroscopy.
Often both the electromagnetic enhancement and the additional chemical enhancement require the addition of special agents. These agents may change the morphology of the surface to produce a size or shape that is more conducive to the electromagnetic SERS effect or they may form mixed complexes with the analyte (cyanide or related species) to produce a product (complex) with a unique electronic absorption. The morphology of SERS active surfaces is crucial for large enhancements. A popular form of a SERS active medium is colloidal particles. These are spherical particles that tend to be so small that they do not aggregate or settle out of solution.
The advantage of colloidal particles is their stability due to lack of aggregation and resistance to settlement due to their small size and continual movement due to Brownian motion. This can also be a disadvantage with respect to SERS. SERS comes largely from the electromagnetic enhancement of light at the particle surfaces. However, this enhancement is strongly dependent on the shape and size of the particle. Spherical particles tend to enhance light at short wavelengths. This can be impractical as laser sources are more common and more intense at longer wavelengths. If the particles become ellipsoidal in shape they exhibit an enhancement at both short wavelengths and longer wavelengths. This arises from the long (long wavelength enhancement) axis and the short (short wavelength enhancement) axis. Furthermore, as the particles become larger, the enhancement is shifted to longer wavelengths.
Up until now, the of detection cyanide and species reactive toward SERS active surfaces has been though direct adsorption to the SERS active surface or through the formation of bonds to a surface bound coating. This is often a sacrificial situation with the actual material responsible for the SERS effect being consumed by the detection method. A more favorable detection method would be to provide a solution species that reacts with the cyanide or related species and then adsorbs to the SERS active surface. This creates a surface species that relates to the cyanide or related species, but it does not consume the SERS active material.
Sometimes a sample contains more than one species (interferers) that can react with an activated SERS surface. At low levels of interferers, it may be possible to spectroscopically distinguish between them and the analyte. However, the amount of surface area available for analysis is limited and the interferers may occupy all sites available for analyte binding. Another situation might be a reaction between the interferers and the activated SERS material to render the SERS active material inactive. In such cases, it may be possible to convert the analyte to a gas and detect it as an adsorbed gas on the activated SERS surface. In this type of analysis, the interferers are left in solution and cannot interact with the spatially separated SERS active surfaces. An example of this type of analysis would be samples containing cyanide and interferers such as thiocyanate or blood metabolites. The sample could be treated with a sufficiently strong acid to produce hydrogen cyanide, but not strong enough to produce volatile sulfur containing species. An accurate assay can be performed if the activated SERS surface is located a distance from the solution such that the hydrogen cyanide can adsorb to the surface, but not the interferers in solution.